Kerainase from fervidobacterium pennavorans DSM 7003

Abstract

PCT No. PCT/EP93/00569 Sec. 371 Date Nov. 2, 1994 Sec. 102(e) Date Nov. 2, 1994 PCT Filed Mar. 12, 1993 PCT Pub. No. WO93/18134 PCT Pub. Date Sep. 16, 1993An enzyme composition containing keratinase is obtained from Fervidobacterium pennavorans DSM 7003. The composition is capable of degradating keratin-containing substrates such as feathers, hair and horn within a few days at between 50 DEG and 105 DEG C. and at a pH of between 4 and 12 under anaerobic conditions. A pH of 10.5 and a temperature of 70 DEG C. or greater are preferred. Dissolving of the substrate can be at least 50% by weight after 24 hours, and in 1 to 4 days the entire substrate can be dissolved. Pretreatment of the substrate at a temperature of 120 DEG C. or greater is not required.

What is claimed is: 
     
       1. An isolated keratinase which has a molecular weight of approximately 200,000 daltons as measured-by gel filtration and which is isolated from Fervidobacterium pennavorans DSM 7003 and has an optimal temperature range between 70° and 90° C. at a pH of 10 and an optimal pH range between 7 and 11 at 80° C . 
     
     
       2. An isolated enzyme composition which contains keratinase and is obtainable from Fervidobacterium pennavorans DSM 7003 and which keratinase has the following characteristics: capable of degrading keratin;   optimum temperature at a pH 10 is in a range of 70° to 90° C.;   optimum pH at 80° C. is in a range of pH 7 to pH 11;   gel filtration exhibits a molecular weight of approximately 200,000 daltons and a determination of molecular weight with an SDS activity gel shows two bands in a range between approximately 200,000 and approximately 330,000;   E 405  value, in comparison to a blank value, after 17 h incubation at 60° C., 0.2 mM substrate in phosphate buffer pH 7 and 0.3 mg/ml raw enzyme extract, measuring stretch 1 cm is > 0.5 for N-succinyl-Phe-pNA, N-succinyl-Ala-Ala-Pro-Phe-pNA and Z-Arg-pNA, between 0.3 and 0.5 for H-Gly-Glu-pNA, between 0.1 and 0.3 for acetyl-Ala-pNA and below 0.1 for benzoyl-DL-Arg-pNA, Z-Gly-Pro-pNA, benzoyl-Lys-pNA, Z-Arg-pNA and acetyl-Tyr-pNA;   inhibitors result in approximately the following residual activities at the indicated following concentrations:   ______________________________________                                    
               Resiudual act.                                             
                          Residual act.                                   
Inhibitor      (1 mM)     (5 mM)                                          
______________________________________                                    
Without        100%       100%                                            
Pefabloc SC (ser.)                                                        
               83%        --                                              
PMSF (ser.)    14%        10%                                             
EDTA (metallo.)                                                           
               93%        85%                                             
Iodoacetate    83%         76%;                                           
(cyst.)                                                                   
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       The thermostabilities are as follows:   at pH 7 and 8.5 is stable over 20 h at 70° C. and 80° C.,   at pH 10, 80° C. and 90° C. after 15 min total loss of activity, and   at 70° C. approximately 40% residual activity after 6h, and after 20 h total loss.   
     
     
       3. The isolated enzyme composition according to claim 2 wherein culturing Fervidobacterium pennavorans DSM 7003 in a tryptone medium induces production of the Keratinase. 
     
     
       4. The isolated enzyme composition according to claim 2 wherein the keratinase has an optimum temperature of approximately 80° C. at a pH of 10. 
     
     
       5. The isolated enzyme composition according to claim 2 wherein the keratinase has an optimum temperature at 80° C. at a pH between 9 and 10.